WO2023004406A2 - Protéines de fusion insuline-fc et méthodes d'utilisation pour traiter le cancer - Google Patents

Protéines de fusion insuline-fc et méthodes d'utilisation pour traiter le cancer Download PDF

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Publication number
WO2023004406A2
WO2023004406A2 PCT/US2022/074036 US2022074036W WO2023004406A2 WO 2023004406 A2 WO2023004406 A2 WO 2023004406A2 US 2022074036 W US2022074036 W US 2022074036W WO 2023004406 A2 WO2023004406 A2 WO 2023004406A2
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fusion protein
insulin
seq
sequence
cells
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PCT/US2022/074036
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English (en)
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WO2023004406A3 (fr
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Todd C. Zion
Thomas M. Lancaster
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Akston Biosciences Corporation
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Publication of WO2023004406A3 publication Critical patent/WO2023004406A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • nucleic acid (cDNA) encoding the fusion protein of SEQ ID NO: 24 comprises the following nucleic acid sequence:
  • the preferred IC50 ratio is less than or equal to 20, or more preferably, the IC50 ratio is less than or equal to 8.
  • the preferred IC50 ratio is less than or equal to 20 or more preferably, the IC50 ratio is less than or equal to 8.
  • Embodiments with lower IC50 ratios demonstrate higher affinities for the insulin receptor than embodiments with higher IC50 ratios.
  • Insulin-Fc fusion proteins that are dimeric with respect to the insulin polypeptide (e.g. two moles of insulin polypeptide chain per mole of insulin-Fc homodimer, e.g.
  • the term “biosynthesis,” “recombinant synthesis,” or “recombinantly made” refers to the process by which an insulin-Fc fusion protein is expressed within a host cell by transfecting the cell with a nucleic acid molecule (e.g., vector) encoding the insulin-Fc fusion protein (e.g., where the entire insulin-Fc fusion protein is encoded by a single nucleic acid molecule).
  • exemplary host cells include mammalian cells, e.g., HEK293 cells or CHO cells. The cells can be cultured using standard methods in the art and the expressed insulin-Fc fusion protein may be harvested and purified from the cell culture using standard methods in the art.
  • in vitro activity or “Fc(gamma) receptor activity” or “Fc(gamma) receptor binding” or “FcRn receptor activity” or “FcRn binding” refers to the affinity with which an insulin-Fc fusion protein binds to the Fc receptor (e.g. Fc(gamma) receptor or FcRn receptor) and is typically measured by the concentration of an insulin-Fc fusion protein that causes the insulin-Fc fusion protein to reach half of its maximum binding (i.e., EC50 value) as measured on an assay (e.g., an enzyme-linked immunosorbent assay (ELISA) assay) using OD 450 nm values as measured on a microplate reader.
  • an assay e.g., an enzyme-linked immunosorbent assay (ELISA) assay
  • the insulin polypeptide of the fusion protein comprises the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 37.
  • the insulin polypeptide of SEQ ID NO: 6 and the insulin polypeptide of SEQ ID NO: 37 include mutations in the B-chain (specifically, B10 is mutated to aspartic acid (D)) and in the A-chain (specifically, A8 is mutated to histidine (H)):
  • fusion proteins e.g., fusion protein described herein
  • a fusion protein described herein is capable of lowering glucose levels (e.g., blood glucose levels) after administration in a subject.
  • glucose lowering activity of the fusion protein is higher than that of an insulin reference standard.
  • the duration of activity of the fusion protein can be measured by a decrease, e.g., a statistically significant decrease, in blood glucose relative to a pre-dose level.
  • the insulin-Fc fusion protein is eluted using a low pH buffer, elution fractions are collected, and the OD280 value of each fraction is recorded. Fractions containing the target insulin-Fc fusion protein are pooled and optionally further filtered using a 0.2 pM membrane filter.
  • the cell line is optionally further subcloned to monoclonality and optionally further selected for high titer insulin-Fc-fusion protein-expressing clones using the method of limiting dilution, a method known to those skilled in the art.
  • production of the insulin-Fc fusion protein is accomplished as described above in growth medium without MSX, or optionally in growth medium containing MSX, to obtain a cell culture supernatant containing the recombinant, CHO- made, insulin-Fc fusion protein.
  • the MSX concentration is optionally increased over time to exert additional selectivity for clones capable of yielding higher product titers.
  • Example 2 Purification of an insulin-Fc fusion protein.
  • Example 5b % Homodimer by size-exclusion chromatography.
  • insulin-Fc fusion proteins of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28 will exhibit a homodimer percentage after Protein A step of Example 2 in excess of 80%.
  • Biotinylated-RHI was diluted in FACS staining medium as a 20x concentration at 10 pg/mL (final 0.5 pg/mL). 50 pL of each serially diluted test compound and 5 pL of 20x Biotin- RHI were added into each well of a V bottom microtiter plate, mixed, and placed on ice. 45 pL of IM-9 cell suspension was then added to each well by multichannel pipette, mixed again gently and incubated on ice for 30 min to allow competitive binding on the insulin receptor (IR) on IM-9 cells.
  • IR insulin receptor
  • FIG. 5 provides Western blots that showed that after 72 hours of treatment, RHI caused observable downregulation of total IR in HCT-116 cells compared to SEQ ID NO: 3 at the higher concentrations tested. Unexpectedly, SEQ ID NO: 1 caused substantial total IR downregulation in HCT-116 cells compared to SEQ ID NO: 3 at all concentrations tested. This is highlighted as 501 in FIG. 5.
  • HCT-116 cells are treated in vitro with either RHI, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 at multiple concentrations (0.05 - 500 nM).
  • Levels of tumor IR, phospho-IR, phospho-IGFIR, phospho-Akt (S473), pan Akt, and Beta (b) Actin expression are measured by Western blot. Tumors are lysed in RIP A buffer, electrophoresed on SDS-PAGE gels, transferred to PVDF membranes using a dry blotting system, and probed for the aforementioned proteins using antibodies from Cell Signaling at 1:1000 along with appropriate secondary antibodies known to those skilled in the art. Blots are imaged (cDigit blot scanner, Licor) and assessed using Image Studio software (Licor).
  • Example 9b Tumor volume in HCT-116 xenograft models in nude mice.
  • mice bearing WM266.4 tumors are randomized into four groups according to Table 11.
  • Example 11 Tumor volume in human breast cancer cell line (MCF-7L) and tamoxifen- resistant breast cancer cell line (MCF-7L TamR) in vivo without fasting.
  • Vehicle and treated group tumor volume is measured before and after the testing and the expected measurements are shown in Table 13, with treatments using SEQ ID NO: 1, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24 are expected to show significant effects both alone and in combination with tamoxifen treatment.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
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  • Diabetes (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Endocrinology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Epidemiology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
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  • Physics & Mathematics (AREA)
  • Emergency Medicine (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente divulgation concerne des compositions de protéines de fusion, par exemple des protéines de fusion à l'insuline-Fc, et leur utilisation pour traiter des cellules cancéreuses et des tumeurs cancéreuses.
PCT/US2022/074036 2021-07-23 2022-07-22 Protéines de fusion insuline-fc et méthodes d'utilisation pour traiter le cancer WO2023004406A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP22846846.8A EP4373861A2 (fr) 2021-07-23 2022-07-22 Protéines de fusion insuline-fc et méthodes d'utilisation pour traiter le cancer

Applications Claiming Priority (2)

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US202163224991P 2021-07-23 2021-07-23
US63/224,991 2021-07-23

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WO2023004406A2 true WO2023004406A2 (fr) 2023-01-26
WO2023004406A3 WO2023004406A3 (fr) 2023-03-02

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Publication number Publication date
WO2023004406A3 (fr) 2023-03-02
EP4373861A2 (fr) 2024-05-29
US11667689B2 (en) 2023-06-06
US20230072260A1 (en) 2023-03-09
US20230272031A1 (en) 2023-08-31

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